The Role of Ambient Air in Preimplantation Toxicology and its

Impact on Clinical Outcomes in In Vitro Fertilization (IVF)

Southwest Embryology Summit

January 4, 2016

Michael P. Magee Kathryn C. Worrilow, Ph.D.

LifeAire Systems

Our Objectives Today •  Discuss the specific role of ambient air

quality in successful preimplantation toxicology and embryogenesis.

•  Discuss the common sources of airborne threats to the laboratory environment - there are as many within the IVF laboratory as there are in the outside source air serving your laboratory and clinical space - which airborne pathogens are critical and which are less important to the process.

Our Objectives Today •  Discuss 3-3-3-4! •  Evaluate current mechanisms of

remediation and their effectiveness •  Recognize what can you do - what

questions can you ask – what solutions exist to assure that your lab design, HVAC system, upstream equipment and protocols are supporting an optimal and consistent laboratory environment?

3 – 3 – 3 – 4 •  3 categories of airborne pathogens

•  3 sources of air •  3 categories of filtration physics

•  4 THM – which led to the identification of a problem and

ultimately a solution

v Outside air serving the HVAC system

v Recirculated air within the space to be protected

v Air provided by the HVAC system

Three Sources of Air

Outside Influences Outside of Your Control

•  Road resurfacing •  Rooftop resurfacing

•  Construction •  Idling engines, exhaust

•  Waste management, restaurant, generator exhaust direction

•  Accidents, tire fires •  Seasonal pollutants

The IVF Laboratory: Common Constituents of Recirculated Air

v  Tissue cultureware §  Styrene §  Toluene §  Acetone §  2-butanone

v  Isopropanol, acetone v  Equipment, material off-gassing v  HVAC / refrigerants / compressed gasses v  CO2, LN2, tri-gas tanks v  Personnel bioburden

HVAC System-Specific Organisms

v Pathogens – viruses, bacteria, fungi v Allergens – bacteria, mold v Toxins – endotoxins, mycotoxins

Operation within an ISO 5 cleanroom - thus removing the variable of air –

•  ISO 5 design incorporating optimal air flow and air dynamics

•  Dedicated AHU and sealed plenum •  30 ACH, + pressure, live monitoring

•  Laminar flow •  > 300 lbs of carbon, > 150 lbs of KMnO4 (Cohen,

Gilligan and Hall) •  ULPA/UVC final filtration (Boone and Higdon)

•  Air flow over critical points of process •  Cleanroom SOPs followed

•  Quarterly certification

How effective was the IVF laboratory cleanroom and HVAC air filtration system

as designed?

+ be

ta/C

linic

al P

regn

ancy

Rat

e (%

+fhb

)

Testing Quarters (TQ) Worrilow et al, 2002, 2008 Worrilow, 2013, 2015

52.4% 54.5%

16.0%

50.0%

37.5%

33.3%

41.6%

13.3%

29.4%

64.7%

55.5%

52.6%

0%

10%

20%

30%

40%

50%

60%

70%

TQ1 TQ2 TQ3 TQ4 TQ5 TQ6 TQ7 TQ8 TQ9 TQ10 TQ11 TQ12

Analysis of CPR, TVOC and Biological Loading within the IVF Laboratory

What did the study of preimplantation toxicology, human embryogenesis, ambient air quality and clinical outcomes tell us? The

study clearly delineated and defined the problem of the variable

of ambient air and the optimal culture environment, and the

specific role that they played in our processes…..it also led to a data

driven solution….

THM #1: IVF does not require the traditional cleanroom environment.

The traditional cleanroom focuses on nonviable particulates, NOT the level of VOCs and viable particulates that must be maintained to optimize the in vitro culture environment for the human

embryo

THM #2: The impact of subtle levels of VOCs on our process and clinical

outcomes

Analysis of CPR & TVOC Loading within the IVF Laboratory

Clin

ical

Pre

gnan

cy R

ate

(%)

Testing Quarters (TQ)

2.7 2.7 2.2 2.2

16.0% 16.0%

37.5% 37.5% 33.3% 33.3% 41.6% 41.6%

13.3% 13.3%

29.4% 29.4% 41.6%

2.7 2.2

16.0%

37.5% 33.3% 41.6%

13.3%

29.4%

0.0

0.5

1.0

1.5

2.0

2.5

0.0

0.5

1.0

1.5

2.0

2.5

TVOC

Levels (Parts Per B

illion)

51.6%

TQ12 0 10

20

30 40 50 60 70 80 90

100

TQ11 TQ10 TQ9 TQ8 TQ7 TQ6 TQ5 TQ4 TQ3 TQ2 TQ1

54.5% 50.0% 52.4%

0.1 0.1 0.1 0.1 0.1

64.7% 55.5%

The presence of ppb VOCs severely compromised clinical outcomes

Worrilow et al, 2002, 2008 Worrilow, 2013, 2015

FAQ: Can’t we simply add more carbon and

KMnO4 to fix the problem?

No.

VOCs Common to the IVF Laboratory

§  Polar VOCs §  Nonpolar VOCs

§  Low MW hydrocarbons §  High MW hydrocarbons

§  Fungal VOCs §  Microbial VOCs §  Biogenetic VOCs

Filtration Physics: Mechanisms of VOC Remediation

•  Physical adsorption •  Chemisorption

•  Oxidation •  Persulfate oxidation

•  Thermal oxidation •  Photo-Fenton oxidation

•  Ultraviolet photocatalytic oxidation (UVPCO) •  Ultraviolet UVV wavelength

•  Molecular sieve •  Transition metal impregnation

•  Fixed bed adsorption •  Surface, contact condensation

“I culture under oil, use tabletop incubators,

time-lapse imaging, etc. VOCs cannot enter my

media or affect my embryos.”

We wish this were true.

If oil soluble, VOCs can easily enter the oil overlay and if water

soluble, the media. Once present in the media or oil, the VOCs are a permanent resident in the culture environment, and thus a permanent threat to the

embryo.

Partition Coefficients

THM #3: The impact of subtle levels of viable

particulates/biologicals on our process and clinical

outcomes

The Impact of Subtle Levels of Airborne Biological Pathogens on

Clinical Pregnancy Rates +

beta

/Clin

ical

Pre

gnan

cy R

ate

(% +

fhb)

(n=54 testing quarters (TQ)) 0

10

20

30

40 50 60 70 80 90

+ beta

53.3% 45.6% 47.1%

17.6%

+ beta CPR

CPR

Loss of power to the ballast boxes supporting the UV lights in our HVAC system coincided with an increase in our clinical loss or

miscarriage rate

Increased biologicals within laboratory air

Worrilow et al, 2002, 2008 Worrilow, 2013, 2015

Filtration Physics: UV v Critical to successful human

embryogenesis and clinical outcome is the intensity, lamp coordinates, level and longevity of the UV output.

v Selection of the right UV source and assurance of maintenance SOP is paramount.

v Not all UV are equal!

THM #4: What HEPA filtration does and does NOT do for our culture

environment

We create the perfect environment for growth…..

IVF laboratory room temperature, humidity and

HEPA/ULPA filter substrate = proliferation of bacterial and viral spores,

mold and biologicals

HEPA/ULPA Final Filter Above IVF Laboratory

Filtration Physics: Viable Particulates UV, HEPA and ULPA

v HEPA and ULPA filtration are designed to remove or capture particles greater than 0.3 microns in diameter at a 99.97 – 99.99, 99.997 – 99.999% effectiveness rating, respectively.

v HEPA and ULPA filtration remediate by “capture.”

Confidential Confidential

v  If air bypass or filter leakage are present and/or if inadequate maintenance and change-outs, the capture rate can drop to 99.94 – 99.95%.

v  Such decrease in efficiency can allow, in the course of a single day, 7.2M particles to penetrate the HEPA filter and contaminate the IVF laboratory and clinical procedure rooms.

v  Effective log kill of viable particulates, bacterial, fungal and viral spores demands the use of the proper UV wavelength, intensity, lamp coordinates, level and longevity of the output.

Filtration Physics: Viable Particulates UV, HEPA and ULPA

+ be

ta/C

linic

al P

regn

ancy

Rat

e (%

+fhb

)

Testing Quarters (TQ) Worrilow et al, 2002, 2008 Worrilow, 2013, 2015

52.4% 54.5%

16.0%

50.0%

37.5%

33.3%

41.6%

13.3%

29.4%

64.7%

55.5%

52.6%

0%

10%

20%

30%

40%

50%

60%

70%

TQ1 TQ2 TQ3 TQ4 TQ5 TQ6 TQ7 TQ8 TQ9 TQ10 TQ11 TQ12

Data Collected Over the 10 Year Study Led to the Identification of the Problem

Identification of the Problem Led to a Comprehensive

Understanding ~ Understanding the source of and impact of subtle levels of VOCs and

airborne biologicals on our processes and defining the optimal in vitro culture environment led to a comprehensive

understanding of the critical nature of the environment that we provide ~

The absence of a solution led to the genesis of the

LifeAire Systems technology.

v System designed to comprehensively KILL and remove all

airborne pathogens: chemical, biological and particulate

ON A SINGLE PASS and produce NO byproducts.

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•  UCSF Medical Center •  Stanford University Medical Center

•  Northwestern University Medical Center •  University of Connecticut Health Center

•  University of Iowa Hospital

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Variation in Protocols ü Controlled O2

ü Tabletop incubators ü Upright CO2 incubators ü Continuous culture ü Sequential culture

ü ICSI, PICSI, PGD, PGS ü Day 5/6 vitrification

ü IVF clinical and laboratory staff

ü Control of ambient air

Shared Variable

Third Party Validation: Alpha Environmental

Fertility and Sterility, Vol. 102; Issue 3 Suppl, p. E91.

Fertility and Sterility, Vol. 102; Issue 3 Suppl, p. E91.

In addition to air purification…..know what you have in your HVAC

system, pop the ceiling tiles and ask questions….lots of

questions……

Critical Components: HVAC System

•  Location of air intake •  Dedicated air handling unit (AHU) •  Water and gas pipelines •  Upstream filters •  Chiller •  Dedicated humidity system •  Proper dehumidification, cooling and

heating equipment •  Comprehensive air purification system

Quality Control Initiatives Towards the Optimal Environment

•  Air quality component, testing and management

•  Air quality over critical points of process, energy efficiency

•  Air testing, comprehensive TAB •  Materials selected for laboratory and clinical

space •  Operational considerations to protect

environment •  Clinical and laboratory staff and work flow

towards the optimal environment

When HVAC Solutions Are Proposed: Ask for Data! Proof of Technology! •  Do they understand the environment that you

are protecting, the vulnerability of the human embryo?

•  Can they assure consistency and performance of your environment?

•  Do they understand the requirements needed by your upstream HVAC equipment?

•  Is the upstream HVAC equipment meeting your criteria?

•  How will maintenance and SOP protocols be followed to assure consistency?

In Summary v The air serving our in vitro culture environment

is dynamic in nature and is influenced by the outside source air, materials used in the laboratory, laboratory protocols, staff, the HVAC design relative to your critical points of process, and all associated HVAC upstream equipment.

v Subtle levels of airborne VOC and biological contaminants can be impactful to successful human embryogenesis and clinical outcomes – proper mechanisms of filtration physics must be used to comprehensively remediate, remove and control the airborne pathogens.

v Using ultraviolet genomic modeling, mathematical modeling and a proprietary engineered molecular media, the LifeAire System comprehensively captures and inactivates all chemical and biological contaminants.

v  It is critical to control the quality of the ambient air serving your in vitro culture environment to optimize successful preimplantation embryogenesis and provide improved levels of patient care.

Success is in the air.